J. Phys. Chem. 1993,97, 9154-9160
9154
Triplet-State Reactions of Hypericin. Time-Resolved Laser Photolysis and Electron Paramagnetic Resonance Spectroscopy Albert Michaeli,t Ayelet Regev,t Yebuda Mazur,* Jehuda Feitelson,? and Haim Levanon**t Department of Physical Chemistry and The Farkas Center for Light-Induced Processes, The Hebrew University of Jerusalem, Jerusalem 91 904, Israel, and Department of Organic Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel Received: May 10, 1993'
Hypericin (Hyp), a naphthodiantrone derivative carrying hydroxyl and methyl substituents, lately attracted much attention because of its possible use a therapeutic agent-especially against retroviral-induced diseases. Here, the excited triplet state of the molecule, 3Hyp, is being studied by laser photolysis and by EPR spectroscopy both in ethanol and in a nematic liquid crystal. It was found that, in addition to its first-order decay (kl = 780 s-1) 3Hyp loses its excitation energy through quenching by ground-state Hyp (k2 = 3.5 X lo7 M-1 s-l 1, by triplet-triplet annihilation (k3 = 2 X lo9 M-l s-l), by energy transfer to another Hyp molecule, by electron transfer to a suitable electron acceptor (duroquinone, ~ E ' I = . 4.5 X lo9 M-l s-I), and by energy transfer to an aromatic porphyrinoid acceptor (porphycene, kEnT = 2.7 X lo9 M-l s-l). The rise and decay of 3Hyp as well as its quenching by various processes were followed by laser photolysis. The energy transfer was studied by laser photolysis, and the electron transfer was followed both by laser photolysis and by EPR. In addition, the EPR measurements enabled us to establish the orientation of 3Hyp, as a guest, in the liquid crystal host, where it was found to exist in the monomeric form. Analysis of magnetophotoselection EPR experiments in ethanol, in which Hyp is illuminated with laser light polarized in parallel and perpendicularly to the magnetic field, showed that a t low concentrations (1VM) the molecule in its monomeric form has its optical transition moment M), a dimer is formed. The angles between the in the molecular plane. At higher concentrations (3.5 X planes of the dimers were estimated to be 25'.
Y
Introduction Hypericin (Hyp) is a naphtodianthrone derivative carrying a number of hydroxyl and methyl substituents (Figure 1). The molecule is a constituent of plants of the genus Hypericum and has been identified as the photoreceptor in the blue-green ciliate Stentor coeruleus.' It was found that in the excited singlet state the pK value of the molecule decreases, and it becomes a fairly strong acid.2 The detrimental effects of Hyp by photodynamic action via the singlet oxygen mechanisms have been reviewed by Song.' Lately, the photosensitized formation of singlet oxygen by Hyp and its reaction inhibiting the action of succinoxidase in mitochondria have been described by Thomas et ala3 The possibility that the photodynamic action of Hyp takes place via a radical mechanism has also been menti~ned.~~s Hyp and its hydroxyl-substituted analogue pseudohypericin currently are receiving much interest because of their possible use as therapeutic agents. Hyp has previously been used as an antidepressant;' however, the main interest now derives from its potential to act at low, nontoxic concentrations as an agent against retroviralinduced diseases. Most intriguing is the possible use of Hyp and pseudohypericinagainst the acquired immunodeficiencysyndrome (AIDS).6 Some of the photophysical properties of Hyp have been determined by Jardon et al.' In addition to its absorption and fluorescence spectra, the triplet-triplet absorption spectrum in solution and in micellar dispersion were measured.8. It was also shown that energy transfer between Hyp and the aromatic compounds anthracene, tetracene, and perylene can take place.8 Here, we describe the triplet state of the sodium salt of hypericin (Hyp) and its reactions by two complementary time-resolved spectroscopies: laser photolysis and electron paramagnetic resonance (EPR) in the CW and pulsed modes. These techniques + The Hebrew University of Jerusalem.
8 The Weizmann Institute. *Abstract published in Aduance ACS Abstracts, August 15, 1993.
t
OH 0
OH
Figure 1. Structure of Hyp. The indicated X and Y axes are the ZFS tensor components fixed in the molecular plane. M is theoptical transition moment, in the molecular plane with 6 = 4 5 O - the angles of M with regard to the ZFS magnetic axes X and Y. @O is the angle between the X axis and the liquid crystal director, L. The direction of L with respect to the ZFS magnetic axes is defined in the text.
were used to characterize the triplet state of Hyp in ethanol and in a nematic liquid crystal (LC) and to follow the electron transfer (ET) from 3Hyp to duroquinone (DQ) and the energy transfer (EnT) from 3Hyp to zinc octaethylporphycene(PC)9 in ethanol.
Experimental Methods The sodium salt of hypericin was prepared and purified according to a procedure described previously.10 DQ (Aldrich Chemical Co.) was recrystallizedfrom petroleum ether and kept in the dark. The liquid crystal (E-7, BDH'1) and spectroscopic grade ethanol (Merck) were used without further purification. Absorption spectra were measured on a diode array HP-8452A spectrophotometer. Zinc octaethylporphycenewas kindly provided by Prof. E. Vogel.9
0022-365419312097-9154$04.00/0 0 1993 American Chemical Society
The Journal of Physical Chemistry, Vol. 97, No. 36, 1993 9155
Triplet-State Reactions of Hypericin Laser photolysis was performed by illuminating the ethanol solutions of Hyp with a Laser Photonics nitrogen-pumped dye laser (LN 1000) at 532 nm. The signal was fed via a Bausch and Lomb 50-cm focal length (f = 4) monochromator and a Hamamatsu R-928 photomultiplier into a Tektronix 2440A oscilloscope and further into a dedicated Olivertti PC for data averaging and data analysis. Solutions were freed of oxygen by the freeze-pumpthaw method in a Teflon-stoppered assembly consisting of the evacuation vessel and a 1-cm optical path cell. All laser photolysis experiments were carried out at room temperature and at concentrations beween 5 X 1 W and 2 X 10-5 M. As shown below, at these concentrations the Hyp in ethanol solution exists in its monomeric form, both in the ground and in the excited states. To check for any permanent changes in the sample absorption, optical spectra were taken before and after each photolysis experiment. Time-resolved EPR measurements were performed by using selectivelaser excitation, both in the CW and in the pulsed Fourier transform (FT) modes. CW-EPR measurements were alsocarried out by employing the magnetophotoselection (MPS) method.‘* Samples of Hyp (10-4 and 3.5 X 10-3 M), dissolved in ethanol, were prepared in Pyrex tubes (4-mm o.d.), degassed by freezepumpthaw cycles, and sealed under vacuum. Samples of Hyp (3.5 X 10-3 M) in the LC (BDH E-7) were prepared by first evaporating the isotropic solvent and then introducing the LC. They were subsequently degassed and sealed. A detailed description of sample preparation and alignment procedure is given elsewhere.I1J3 In the direct detection mode, the sample in the microwave cavity was photoexcited at 532 nm by the second harmonic of a Nd:YAG (Quanta Ray, DCR- 1A) laser of 10-ns-pulse duration (20 mJ/pulse) with a repetition rate of 10 Hz. CW spectra (Varian E-12) at different specified delay times after the laser pulse, Td,wereobtained from the transverse magnetization, My(t), decay curves. A description of light excitation, signal detection, and the real time data acquisition has been given previously.l3 Triplet line shape analysis was carried out on a CCI Power 6/32 minicomputer. MPS experiments were performed by utilizing two polarizedlight excitations, Le., where the dominant electric field components are either vertically ( E I B ) or horizontally polarized (EIIB) with respect to the external magnetic field.12J4 Changing from the initial vertical polarization to the horizontal one was carried out by utilizing a set of one mirror and four prisms, located on different ~ 1 a n e s . lThe ~ final polarization in front of the EPR cavity was verified by a commercial polarizer, maintaining the same intensity for the two polarizations. For FT-EPR measurements, we used a pulsed EPR spectrometer (Bruker ESP 380) interfaced to a pulsed Nd:YAG laser (Continuum, Model 661-2D) and providing light pulses (20 Hz, 12-nsduration,-2OmJ/pulse) at X = 532nm. Freeinduction decay (FID) signals were detected (using 24-11s microwavepulses) at selected delay times, Td. The minimum time between two successive time-dependent FT-EPR spectra was 10 ns and was determined by a digital oscilloscope (Lecroy, 9400A). The FID dead time was about 130 ns in these experiments. A linear prediction singular value decomposition (LPSVD) routinel5 was applied for reconstruction of the FIDs within the dead time. Temperature was maintained by a nitrogen flow system at 120 Kfor triplet-statedetection and -240K for the ET measurements.
-
-
Results
6
a
-
-€ i: 4 X
w“
0.2 0.1
-0.2
-‘ -
1
tl
2-
1.5 -
2
t
2 x
wc
10.5 7 0
320
220
520
620
’
10
Unm) Figure 2. Triplet absorption spectrum of hypericin: (a) ground-state absorption, (b) measured- AOD spectrum, (c) corrected T-T absorption spectrum. The following processes of photoexcited Hyp were studied: hv
klsc
Hyp-, ‘Hyp
’Hyp
excitation and ISC
(1)
ki
’Hyp
-
Hyp
first-order decay
(2)
ki
3HYP + HYP
3Hyp 3Hyp
HYP + HYP quenching by ground-state molecules (3)
+ 3Hyp
+ Hyp
-
k3
kex
Hyp
‘Hyp
+ Hyp
+ 3Hyp
T-T annihilation (4)
triplet energy hopping ( 5 )
In the presence of DQ, electron transfer takes place
+
+ ker
+
3Hyp DQ Hypo+ DQ’- electron transfer (6) and in the presence of PC, energy transfer was observed kanr
The absorption spectra of Hyp in ethanol in the concentration range of 5 X 10-6 to 3.5 X 10-3 M were found to be identical both in the location and shapes of the peaks, as well as in the intensity ratios of the various absorption maxima. Hence, we assume that over this concentration range the molecule in its ground state does not form dimers in ethanol.
420
+
3Hyp PC Hyp 3PC energy transfer (7) Triplet State. 1 . Laser Photolysis. The uncorrected and the corrected triplet-triplet absorption spectra of Hyp, excited at 532 nmin ethanol, are shown in Figure 2. The intersystem crossing (ISC) yield was determined by the procedure of Medinger and Wilkinson.’* Fluorescence and triplet-triplet absorption of Hyp
9156
1
3 C a
Michaeli et al.
The Journal of Physical Chemistry, Vol. 97, No. 36, 1993
..
1
0.75
.3
8
.4
13
2 0.5
v
b
:'a
2 0.25
I
I
I
0.450 0.675 0.900 Time (msec) Figure 3. Decay of hypericin triplet at different Hyp concentrations: (a) [Hyp] = 9.6 X 1od M, (b) [Hyp] = 2.4 X l k 5 M, (c) [Hyp] = 4.8 X l k J M. Insert shows kl' dependence on [Hyp] (q8). 0.225
0
were measured in the presence of ethyl iodide. A value of q& = 0.52 f 0.05 was obtained. In Figure 3, we show the triplet decay curves of Hyp in ethanol in the absence of oxygen at room temperature (295 K). The decay curve was analyzed by solving the differential equation for first- and second-order processes 4 3 H Y P l l d t = k,T3HYPl + M3HYPlL3HYPl which integrates to i3HYPl, = (kl'[3HYPlo)/KexP
+ k,[3HYPl,)
k" l'
-
k,[3HYPl,) (8) where the subscript 0 indicates time zero. The first-order rate constant kl' showed a linear dependence on the Hyp ground-state concentration (cf. inset of Figure 3). We, therefore, assume that the 'Hyp disappears by the quenching I
w
'
I
Y1
'
x1
I
'
xz
I
'
YZ
process (3) in addition to its nonradiative decay and the triplettriplet annihilation reaction (4). We obtain kl' = kl + kz[Hyp] with a first-order decay constant, when extrapolated to zero Hyp concentration, of 780 f 150 s-l, similar to the value reported by Jardon.' The pseudo-first-order constant kl' for the quenching of 3Hyp by ground-state Hyp yields kz = (3.5 f 1.0) X lo7 M-l s-I. Having kl', the kinetic curves can be simulated according to eq 8, resulting in the rate constant for the triplet-triplet annihilation, which is within the diffusion limit, Le., k3 = (2.0 f 0.4) X 109 M-1 s-l, again similar to the reported data.7 2. EPR Spectra. Time-resolved EPR spectra were measured by the direct detection CW and FT methods. The CW experiments were carried out in a nematic liquid crystal (LC) and in an ethanol glass at low temperature, and the FT experiments were carried out at high temperature in liquid-phase ethanol. a. CW Measurements: Nematic Liquid Crystal. To relate the principal axes of the ZFS tensor to the optical transition moment, M, of Hyp, we first examined the triplet EPR line shape of HYP in the nematic LC. The CW-EPR triplet spectra of Hyp at parallel (LIIB) and perpendicular ( L I B ) orientations of the LC director, L, relative to the external magnetic field, B, are shown in Figure 4. The nonderivative line shapes reflect spinpolarized spectra that exhibit an a,e,a,e,a,e pattern (from low to high field), where a stands for absorption and e for emission. The noticeable intensity attenuation of the four outermost lines (Y and Z canonical orientations) in the parallel configuration of L (Figure 4b) suggests that Hyp orients itself in the LC such that LIIX. The spectral width at the L I B is comparable to that obtained in the ethanol glass matrix at low concentration (Figure Sa). Therefore, we attribute the LC spectra to the monomeric species, as will be detailed below. The almost complete disappearance of the outermost canonical orientations in the LJIB spectrum indicates an apparent planar structure of the Hyp monomer, with its Z magnetic axis out-of-plane. It is consistent with the structure in which the spectral width at the L I B orientation is larger than that obtained for the LllB orientation. This line shape analysis corresponds to a PT* transition, where the optical transition moment, M, is perpendicular to the dipolar Z axis and, hence, lies in the molecular plane. The EPR line shape for a partially oriented triplet in the LC can be expressed quantitatively in terms of the density matrix, I
I
22
21
A
b
a
x
x
'I
2765
3265
3765
2765
'
I
Y1
'
I
XI
'
x2
I
'
I
YZ
21
A: 3265
3765
Magnetic Field (Gauss) Figure 4. CW-EPR triplet spectra of Hyp in E-7 liquid crystal (3.6 X lk3M) at the perpendicular, a, and parallel, b, configurations. Pulsed laser excitation at 532 nm, 12 mJ/pulse. All spectra were taken 500 ns after the laser pulse at 133 K and 40-mW microwave power. The arrows indicate the canonical orientation. The smooth curves are simulations obtained by use of eq 9 and the parameters listed in Table I.
The Journal of Physical Chemistry, Vol. 97, No. 36, 1993 9157
Triplet-State Reactions of Hypericin
1 J J
J
J J
J
b
I E II B
1
2765
3265
3765
2765
3265
3765
Magnetic Field (Gauss)
I
2765
l
22
l
Y1
l
'
x1
l
3265
'
x2
l
i
Y2
l
21
3765
2765
3265
3765
Magnetic Field (Gauss) Figure 5. Magnetophotoselection (MPS) time-resolved triplet EPR spectra of Hyp, randomly oriented in an ethanol glass: (a,b) low concentration, lo" M (monomer); (c,d) high concentration, 3.6 X l k 3 M (excimer). Pulsed laser excitation (EIIB and E I B ) at 532 nm, 12 mJ/pulse. All sptctra were taken 500 ns after the laser at 133 K and at 40-mW microwave power. The arrows indicate the canonical orientations. The smoothed curves are simulations obtained by employing eq 1 1 and the parameters listed in Table I.
by the imaginary part of the magnetic susceptibility, x":13J4 x"(B,t) a
where 0 and 4 are the angles between the magnetic field, B,and the principal axis system of the magnetic dipolar tensor. The functions f(6") and f(4) reflect the distributions of the guest compound about the director, L. The density matrix element, pij(O,$,f), connects the i j triplet spin levels. The summation is over the two possible triplet transitions. Best-fit triplet line shape analysis of the experimental CWEPR data according to eq 9 results in the triplet ZFS parameters, the triplet singlet relative population rates (Table I), and the anisotropic distribution in the LC matrices. The analysis shows
-
TABLE I: Triplet-State Parameters of Hypericin Oriented in Different Matrices solvent I* IW" A,:A,:A, ethanol (monomer) ethanol (excimer) E-7 (monomer) 0
336 336 321
53 53 56
0.01.0:0.84 0.01.0:0.38 0.15:1.00.70
x 10" cm-1 (estimated error f 5).
that the director is close (loo) to the X dipolar axis. Thus, it is reasonable to assume the X as the longest dimension of the molecular plane (Figure 1). b. CW Measurements: Ethanol. Time-resolved EPR experiments using the MPS method1*J4were carried out at low (lW M) and high (3.5 X 10-3 M) concentrations in the ethanol glass. The spectra are shown in Figure 5a,b and 5c,d, respectively. In the MPS method, the triplet spectra of Hyp were taken with the
9158
The Journal of Physical Chemistry, Vol. 97, No. 36, 1993
Michaeli et al.
electric field of the laser polarized either vertically or horizontally, with respect to the magnetic field, i.e., E I B (Figure 5a,c) and EllB (Figure 5b,d). At low concentration, the triplet line shapes exhibit spin polarization similar to the LC spectra, i.e., an a,e,a,e,a,e pattern (from low to high field) both in the vertical and horizontal configurations. However, the relative intensities of the signals in the two configurations differ from each other, as expected in a MPS experiment. The intensity of a given triplet transition, corresponding to a canonical magnetic axis, along the optical transition moment will decrease on going from horizontal to vertical light polarization. Thus, the degree of polarization, Ri, along each canonical axis, i , is defined by12b
18 I
(10) Ri = [(Ill -zA)/(Ili + Il)li where Zll and ZI are the EPR signal intensities for the vertical or horizontal light polarization. A positive value of R, implies that the corresponding i axis is close to the transition moment M. Applying eq 10 to the spectra of Figure 5a,b one finds a negative value of R, for the outer pair of lines. Qualitatively, it follows that the transition moment, M should be perpendicular to the dipolar Z axis. The specific angles between M and the dipolar axes are obtained from the line shape simulations. The EPR line shape for static randomly oriented triplets, in terms of the density matrix formalism, is given by modifying eq 9:13,14
0
where D(B,d,y) is the distribution function for the ensemble of triplets excited by the polarized light, y is the angle between the optical transition moment M and the out-of-plane magnetic axis, and 6 is the angle between the in-plane principal axis and the projection of M onto the molecular plane. The other terms in eq 11weredescribed above (eq 9). Lineshapesimulations according to eq 11 result in the ZFS parameters D and E , the relative triplet population rates and the location of the optical transition moment in the molecular frame (Table I). Indeed, the results show that M is located in the molecular plane with an angle of 45’ from the X or Y axes (Figure 1). The triplet spectra of Hyp in ethanol glass a t high concentration (3.5 X 10-3M) are shown in Figure 5c,d. The line shapes of these spectra differ markedly from those obtained at the low concentration. Although ground-state dimers are excluded a t room temperature by absorption spectra measurements, the variation in the EPR line shapes at 133 K is best interpreted in terms of two site triplet exciton hopping within a dimer. In the case of a triplet exciton, the line shape analysis must take into account the relation between the systems of principal axes of the two interacting monomers (X,Y,Z and X’,Y’,Z?. This relation is defined by a Euler rotation matrix.16 By denoting the two sites of the exciton as A and B, one finds the contribution of the hopping process to the line shape is given by1’
+
= prjA pi; (12) Thus, in addition to the triplet magnetic parameters, the line shape analysis results in the relative orientation of the two monomer units and a value for the triplet energy hopping rate constant between the two components of the excited dimer, i.e., k , in the range 3-9 X lo7 s-I (eq 12). The three-dimensional structure of the excited dimer is also established via this analysis, namely,a= ISo,@=5’,y =25’,wherea,B,andyaretheEuler rotation angles transforming the X,Y,Z,system into the X’,Y’, 2’ frame. These values imply a near face-to-face configuration with an angle of 25’ between the planes of the subunits. 3. Electron Transfer. The Hyp triplet decay in ethanol was measured in deoxygenated solution by triplet absorption following laser excitation in the absence and in the presence of DQ in the p JJ . . E p.PB Y
I
I
10- ./”
I
I
1
I
I
0.5
1
1.5 [MI x io4
2
2.5
3
Figure 6. Stern-Volmer plot of 3Hyp decay in presence of DQ (full circles are electron transfer) and in the presence of PC (open circles are energy transfer). Insert shows decay curves of ’Hyp in the presence of M,(3) [DQ] = DQ: (1) [DQ] = 7 X 1V M,(2) [DQ] = 1.2 X 2.5 X 10-5 M.
2 X le5to 2 X 10-4 M range. Given a 3Hyp concentration of -2 X 10-6 M at our laser intensity, a pseudo-first-order decay can be assumed at the above DQ concentrations. The SternVolmer plot of the reciprocal decay times 1/70 - 1/7 against DQ concentration yields a quenching rate constant of 4.5 X 109 M-l s-l, meaning that the electron transfer is an encounter-limited process, Figure 6 . The radical ion, DQ’-, was observed by FTEPR measurements (see below). Electron transfer between the triplet state of Hyp (10-4 M) and DQ ( M) in ethanol was also monitored by time-resolved pulsed EPR spectroscopy. The FT-EPR spectra obtained by photoexcitation of Hyp in the presence of DQ are shown in Figure
I. Spin polarization effects observed in the EPR spectra of the transient radicals stem from two major mechanisms: the triplet mechanism (TM) and the radical pair mechanism (RPM). The efficiency of T M is determined by the selectivity of the triplet singlet ISC rates to the three triplet sublevels and by the competition between the electron-transfer rate, ET, and the spin lattice relaxation rate, T 1 ~ - lTo . observe TM, T I Tshould obey ET > T I T . -T~M creates a net polarizationmM, where the two radicals have the same polarization phase (absorption or emission). The value of PTM is given by19
-
(13) = kE+T/(kET + TIT-’) where MT is the total polarization of a triplet molecule, and its magnitude ig given by20 PTM
Mi
-[D(A,
+ A, - 2A,) + 3E(A, - A,)]
(14) where D and E are the zero-field splitting parameters and A, ( i = x , y, z ) are the rate constants for the ISC into the different triplet substrates. Assuming that D > 0 and inserting the value of A,, calculated by the line shape analysis (Table I), one obtains a positive value for MT, Indeed, inspection of Figure 7 reveals that for delay times ‘T,J < 1 ps the spectra of DQ’- exhibit an absorption pattern, implying that the T M dominates. However, a t delay times 7 4 > 1 ~ sthe , RPM starts to take over and becomes the dominant process. Time-resolved FT-EPR spectra of the anion radical, DQ-, in the frequency domain exhibit an absorptionemission(a/e) pattern, while in the field domain an e/a pattern isobtained. This multiplet effect is typical of the S-To mechanism which applies to any radical pair obeying the Buckley-McLauchlan theorem.21 This theorem states that the EPR line intensity of the hyperfine component of one radical is zero at the center of the spectrum of the other radical and vice versa (at the point of zero intensity, N
Triplet-State Reactions of Hypericin
The Journal of Physical Chemistry, Vol. 97, No. 36, 1993 9159
300 ns
30 ps
150 ns
9 cts
80 ns
24 ns
-40
0 MHZ
40
-40
0
40
MHZ
Figure 7. FT-EPR spectra of DQ'- (lP3M) in ethanol a t 243 K, with Hyp (1VM) as electron donor, at different delay times between light excitation and microwave detection.
the spectra change phase). The multiplet effect implies that the mixing parameterZZis determined by hyperfine energy terms; that is, Agis small. Actually, avalue of Ag= 0.0005 was estimated from the spectra (cf. Figure 7). For a S-TOmixing and assuming J 20 ps, the polarized spectrum of DQ'- changes into an equilibrated one due to spin lattice relaxation. As in previous s t u d i e ~ ,the ~ . ~rate ~ parameters of ET and spin relaxation were determined by a semiqualitative analysis by studying the mf = 0 and mf = f l hyperfine components of the FT-EPR spectra. The time evolution of the central hyperfine component, Zc = Z(mf=O), and the intensity difference between the two adjacent components of the central line, I A = I(ml=+l) - Z(mf=-l), should differentiate, to a good approximation, between the TM and RPM. Following the treatment of polarized DQ'- due to the ET reaction between an excited precursor and a quinone (eq 6 ) , the time dependence of the signal intensities was fitted by the functionZ3
p = Aj[exp(-kjt) - exp(-t/ T,)]
(15)
where p denotes the polarization in terms of the 1, or ZA terms, j denotes the appropriate TM or RPM values, kj-1 is the rise time by TM or RPM, A, is the relative amplitude, and T1 is the spin lattice relaxation time of DQ'-. To obtain a good fit with the data, it was necessary to take into account the Boltzmann e q ~ i l i b r i u m .The ~ ~ rate constant kRpM equals keT, while kTM = keT + TIT+,where TITis the spin lattice relaxation time of 3Hyp. The time evolution of the IC and IAof DQ'- is shown in Figure 8, and the best-fit analysis results are the pseudo-first-order rate ET = 5 X lo8 M-l s-l and the spin lattice relaxation times TIT = 26 ns and T1 = 9.1 ps. The EPR experiments yield a lower value for the electrontransfer rate constant than the laser photolysis data. This can be expected from the temperature difference between the two experiments. The calculated spin lattice relaxation time of D Q -
io-' 10 10-1 1 10 100 Time (ps) Time (ps) Figure 8. Time dependence of TM (a) and RPM (b) contributions to the CIDEP effect of DQ'- produced by 3Hyp. The TM and RPM polarizations were determined as described in the text, the lines were calculated by use of eq 15, and the best-fit parameters are given in the text.
10-3
is in good agreement with values of T1 for DQ*- obtained with other triplet precursor^.^^^^ 4. Energy Transfer from Hypericin to Porphycene. In addition to the above electron transfer, Hyp can also transfer its triplet energy to porphycene (PC) in solution. Hyp in ethanol at a concentration of 3 X lV5M was excited by the dye laser at 532 nm. Its molar absorption coefficient at this wavelength is e = 16000 M-l cm-1 (Figure 2). The corresponding value for PC is 1700 M-I cm-l.9 The PC concentrations in the deoxygenated solutionswere5 X 10-6,l.l X lV5,and 1.8 X l e 5 M . Ittherefore absorbs very little at the above wavelength. The data were, nevertheless, corrected for the dirct absorption by PC. The appearance of the PC triplet and its rise time as well as its subsequent decay were monitored by laser photolysis (Figure 9). The rise times for the above PC concentrations lie between 18 and 70 ps, similar to the decay time of 3Hyp. The Stern-Volmer plots (Figure 6b) yield an energy-transfer rate of k@nT = 2.7 X 109 M-1 s-1, Le., an encounter-limited energy transfer. To conclude, it is seen that the laser photolysis and EPR spectroscopymeasurements complement each other in providing together a more completedescription of the triplet-state properties
9160 The Journal of Physical Chemistry, Vol. 97, No. 36, 1993
0.0425
n
0 d
0
-0.0425
1
9, :'
bI
Y
of Hyp. The variety of diffusion-limited triplet-state processes of Hyp might explain its high reactivity in biological systems.
Acknowledgment. We are grateful to Mrs. Assia Berman for her assistance in the analysis of the FT-EPR data and to Prof. E. Vogel for the zinc octaethylporphycene. The Farkas Research Center is supported by the Bundesministeriumfiir die Forschung und Technologieand the Minerva Gesellschaft fiir die Forschung GmbH, FRG. This work was supported by a DFG grant (Sfb 337). This work is in partial fulfillment of the requirements for a Ph.D. degree (A.M.) at the Hebrew University of Jerusalem. References and Notes (1) Duran, N.; Song, P. S.Phorochem. Phorobiol. 1986, 43, 677. (2) Walker, E. B.; Lee, T.Y.; Song,P. S. Biochem. Biophys. Acta, 1979, 587, 129.
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